A set of precise programs of gene expression control many complex biological processes, such as cell growth, differentiation, and development. An elucidation of the mechanisms by which the normal controls are aborted, as in various neoplasms, birth defects, and other diseases, will therefore require a basic understanding of the regulation of gene expression. In general, regulation of RNA polymerase II (pol II) transcription of a gene controls its expression. A complex set of DNA sequences and proteins are required for initiation of transcription at pol II promoters. The promoter sequences can be divided into: 1) an initiation region, extending from -35 to +20 (where +1 is the initiation site), 2) an immediate upstream region, extending from about -110 to -50, and 3) enhancer sequences, positioned at widely varying distances. The mechanisms by which the latter two classes of sequences activate transcription are particularly intriguing and unclear, due to their effects bidirectionally and at varying distance. The objective of this proposal is to define how the immediate upstream region of pol II promoters can stimulate the initiation of transcription. The characterization of both DNA and protein components is intended. Activity of these components will be measured by effects both on transcription, in vivo and in vitro, and on protein-DNA interactions, assayed in part by a rapid nitrocellulose filter binding assay. The focus of the proposed project is the SV40 early and late promoters. These promoters are uniquely suited to such studies because: 1) transcription in vitro and in vivo is strongly dependent on their shared immediate upstream region, 2) transcription in vitro is also strongly dependent on a specific cellular factor, which seems to function through the upstream DNA sequences, and 3) an assay which monitors specific binding of a cellular factor to the upstream sequences has been developed. The specific aims of the proposed project are 1) to determine if the SV40 immediate upstream sequences can stimulate transcription in varying positions and orientations relative to a heterologous initiation site, 2) to isolate and mutagenize the minimal active sequences of the SV40 upstream region, 3) to purify the cellular protein(s) which specifically binds the SV40 upstream sequences, 4) to determine whether the DNA-binding protein(s) is involved in transcription, 5) to characterize the interactions between the DNA-binding protein(s) and SV40 DNA, and 6) to identify other viral and/or cellular promoters which interact with the protein(s).